Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains a major public health problem worldwide. Ty21a is a licensed live-attenuated oral typhoid vaccine that has shown efficacy; however, it is modestly immunogenic and requires multiple doses. Because of the appearance of multiple antibiotic resistance S. Typhi strains there is renewed urgency for developing more effective typhoid vaccines. However, a major obstacle for the development of novel, more immunogenic, typhoid vaccines remains the lack of knowledge of the immunological correlates of protection (CoP) from S. Typhi infection in humans. This is due, in large measure, to the extreme difficulties associated with performing challenge studies with wild-type (wt) S. Typhi in volunteers and the lack of a small animal model that faithfully recapitulates human disease. Dr. Pollard (Oxford, UK) has recently reestablished, in collaboration with the CVD in Baltimore, the challenge model originally pioneered at UMB in the 1960's in which subjects were orally challenged with wt S. Typhi. Moreover, a follow up study in which subjects are being vaccinated with the M01ZH09 attenuated typhoid vaccine candidate and will be challenged with wt S. Typhi is already underway. These studies provide a truly unique opportunity to uncover the immunological CoP from S. Typhi infection and dramatically accelerate the development of novel, more effective vaccines. The immune responses to S. Typhi in humans are complex, involving antibodies (Ab) and cell-mediated immunity (CMI). We hypothesize that the induction of potent and sustained CMI and humoral immunity at the local and systemic levels is critical for the development of effective typhoid vaccines. Studies conducted by the PI over the past 20 years, largely supported by this R01, have shown that immunization with attenuated typhoid vaccines elicits memory B cells (BM), are well as potent CD4+ and CD8+ CMI responses, including multifunctional Tc17 memory T cells (TM). We propose to use specimens isolated from subjects (1) challenged with wt S. Typhi, (2) immunized with M01ZH09 or Ty21a followed by wt S. Typhi challenge and (3) immunized with one (non-protective) or four (protective) doses of Ty21a, to: (1) test the hypothesis that the kinetics of appearance, magnitude, characteristics, persistence and homing potential of defined specific multifunctional memory (TEM, TEMRA, TCM) and effector T cell (Teff) subsets and BM in circulation after challenge with wild-type S. Typhi predic whether the subjects will develop typhoid fever, (2) validate the findings in Aim 1 by evaluating in subjects immunized with attenuated S. Typhi strains whether similar responses can predict protection from the development of typhoid fever upon challenge with wt S. Typhi, and (3) test the hypothesis that protective MHC class II-, class Ia- and HLA-E-restricted CMI against S. Typhi is determined by a restricted set of epitopes derived from S. Typhi proteins.